Electrophoresis is a separation process well known in the art (C. F. Simpson, Electrophoretic Techniques, Academic Press, New York (1983)) and is achieved by passing an electric current through a porous matrix to which the compounds to be separated have been applied. The compounds migrate through the matrix at a rate that is dependent upon the size and/or charge to mass ratio of the compound and the strength of the electric field that is applied to the matrix. This technique can be used to separate small compounds including carbohydrates and amino acids, as well as large macromolecules such as polysaccharides, proteins, DNA, and RNA (M. Dubois et al., Analytical Chemistry 28, 350-356, (1956), C. Tsai et al., Analytical Biochemistry 227, 115-119 (1982), U. K. Laemmli, Nature, 227, 680-685 (1970), F. Sanger et al., Proc. Nat. Acad. Sci. U.S.A. 74. 5463-5467 (1977), P. S. Thomas, Proc. Nat. Acad. Sci. 77, 5201-5212 (1980)). Various matrices can be used for the separation of compounds by electrophoresis including paper, starch, agarose, and polyacrylamide.
Electrophoretic separation generally includes the following steps: the separation gel or matrix is equilibrated with a suitable electrophoresis buffer that will be used during the electrophoresis process, each end of the equilibrated matrix is placed in contact with a reservoir also containing a buffer, a positive electrical lead is placed in one of the reservoirs while the second reservoir has a negative lead attached, the samples are applied to the matrix, and an electric field is generated (C. F. Simpson, Electrophoretic Techniques, Academic Press, New York (1983)). Once the electrophoretic separation is completed, the separated compounds can be visualized by a variety of different methods (M. Dubois et al., Analytical Chemistry 18, 350-356 (1956), C. Tsai et al., Analytical Biochemistry 119, 115-119 (1982), S. M. Hassur et al., Anal. Biochem. 41, 51 (1971)).
In many electrophoresis separation systems, the samples are applied to a small well which is formed in the matrix to provide a guide for loading the sample. This sample well is typically submerged with the electrophoresis buffer. A suitable loading solution therefore needs to have a density that is greater than that of the electrophoresis buffer. Other components of a suitable loading solution include reagents that stabilize or denature the sample. To meet these needs, substances such as formamide, glycerol, ficoll, sucrose, SDS, or urea are typically added in the loading solution. In addition, it is desirable to include as a component of a loading solution, a dye that facilitates visualization of the sample during the loading process. The addition of a colored dye to the loading solution allows easy visualization of the solution as it is applied to the sample well. Among the dyes that have been used in loading solutions, include bromophenol blue and xylene cyananol (J. Sanbrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 6-12 (1989)). These dyes migrate in the electric field during electrophoresis and can be used to estimate the distance the compounds have migrated through the matrix.
Recently, systems have been developed that rely on detection of the compounds as they migrate through the matrix. These systems have fixed detection zone(s) that typically employ the use of photomultiplier tubes to detect light (J. M. Prober et al., Science, Vol. 238, 336-341 (1978)). Dyes known in the art (J. Sanbrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 6-12 (1989)) can interfere with the detection of compounds that migrate through the detection zone at or near the same point in time as the dye. Thus, there is need for dyes which facilitate the loading of samples onto a matrix but which do not interfere with the detection of compounds.